Late preconditioning (PC) provides sustained and powerful protection against both reversible and irreversible ischemia/reperfusion injury. The underlying mechanisms of late PC have been extensively investigated in the hope of identifying new approaches to protect the ischemic myocardium. Thus far, however, most studies have been undertaken in animal models in which ischemia was imposed in the absence of other disease processes. Since pathologic conditions may interfere with the biochemical pathways that underlie late PC, the PC response is likely to be modified by systemic diseases. Therefore, there is an urgent need to examine the PC response in the presence of concurrent, clinically relevant abnormalities such as hypercholesterolemia (HC). It has been well documented that HC impairs nitric oxide (NO) formation and that NO plays a critical role in late PC, acting initially as a trigger and subsequently as a mediator of this phenomenon. The overall objective of this proposal is to investigate the late PC response in the presence of HC. Our fundamental hypothesis is that the cardioprotective effects of late PC are abrogated by HC, and that this is caused by NO insufficiency both at the level of trigger (immediately after the PC stimulus) and at the level of mediator (24 h later). We will first verify that both the anti-stunning and the anti-infarct effects of late PC are abolished in chronically-instrumented rabbits with HC; the dose dependent effects of HC will be established. We will then examine the effect of HC on the generation of NO that triggers the signal transduction events that initiate the late PC response. Furthermore, we will determine whether HC blocks formation of NO from the inducible NOS, which mediates the cardioprotection of late PC. The role of ADMA disregulation and DDAH activity in impairing eNOS-dependent NO generation during HC will be investigated. The role of tetrahydrobiopterin (BH4) deficiency in impairing the function of NO as a mediator of late PC will also be established. The ability of supplemental L-arginine, NO donors, and exogenous BH4 to reinstate the late PC response in the presence of HC will be investigated. This study will thoroughly test the concept that HC abrogates late PC by impairing the role of NO, and will interrogate the underlying mechanisms. The results may pave the way for therapeutic strategies aimed at restoring the late PC response in the clinically prevalent condition of HC. [unreadable] [unreadable]